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Complex in vivo regulation of matrix metalloprotease (MMP)-2 and MMP-9 makes measuring their activity challenging. MMPs are synthesized as inactive zymogens, and must be enzymatically activated by hydrolytic cleavage of a pro-peptide domain. Activity of the resulting enzyme is further regulated by endogenous tissue inhibitor of metalloproteases (TIMPs).
Measuring MMP protein abundance and localization by classic methods such as western blot, ELISA or IHC is not sufficient to accurately describe protein activity in vivo1. Multiple methods have been developed to look at MMP-2 and MMP-9 activity. Find which assay is most appropriate to answer your research questions.
Suitable as a crude measurement2 of maximum activity in a small number of biological and clinical samples in vitro
Use for high-throughput, quantitative data in vitro
Use to obtain non-quantitative, localization data
Use when you need 3D, real-time patterns of activity in vivo
Compatible sample types: conditioned media, tissue extracts.
How it works: proteins are separated by electrophoresis, using a non-reducing SDS-PAGE gel that is embedded with gelatin. After separation, SDS is removed from the gel, and the gel is submerged in a solution that contains essential cofactors required for enzyme activity. MMPs within the gel digest the gelatin, resulting in clear bands on a dark blue background after staining with Coomassie.
Advantages: gel zymography is an easy and low cost method that provides highly sensitive detection of gelatinase activity3. The activities of different gelatinase enzymes, as well as their active and zymogen forms can be distinguished based on their migration through the gel, especially if 2D gel electrophoresis is used4.
Considerations: this technique is suitable for obtaining qualitative or semi-quantitative data, but getting reliable quantitative data with this method is challenging2. Gel zymography is laborious for handling multiple samples, and not suited to high-throughput analysis.
MMP regulating factors such as TIMPs dissociate from MMP-2 and MMP-9 during electrophoresis, therefore gel zymography does not give an accurate measure of in vivo activity, but rather estimates the maximum potential activity in the tissue.
When to use: use as a crude measurement of maximum activity2 in a small number of biological and clinical samples.
How we can help: gelatin zymography protocol.
Compatible sample types: conditioned media, tissue extracts.
How it works: substrate assays involve the use of artificial MMP-9 substrate peptides that contain a fluorophore at one end, and a quencher at the other. In their intact state, fluorescence is minimized due to fluorescent quenching.
After combining peptides with liquid samples, the peptides are hydrolyzed by MMP-9 and fluorescence increases as the quencher diffuses away from the fluorophore.
Similar non-fluorescent assays have also been described that use biotinylated gelatin and succinylated gelatin5,6.
Advantages: substrate-based assays provide highly sensitive, quantitative data, and can be combined with ELISA to calculate specific enzyme activity7. The assay is rapid and can be adapted to a high-throughput format.
Considerations: tissue homogenization may lead to artificial mixing of enzymes and inhibitors, generating results that do not report true in vivo activity8. Additionally, the use of artificial peptides may result in erroneous results if structural differences between the peptide and the endogenous substrate affect enzyme kinetics. However, triple helical substrates that more closely mimic the natural substrate structure have also been developed9.
Substrate specificities for MMP-9 and MMP-2 are similar, making it hard to differentiate activity of the individual enzymes. This issue can be avoided by including a step to isolate either MMP-9 or MMP-2 using antibodies10.
When to use: use to obtain high-throughput, quantitative data.
How we can help: MMP activity assay (Fluorometric green).
Compatible sample types: frozen tissue sections or ethanol- and zinc-fixed paraffin-embedded tissue sections1.
How it works: tissue sections are bathed with a fluorescent substrate peptide. As the fluorescent substrate is hydrolyzed by MMP-9 present in the tissue section, loss of fluorescence is seen as black holes on a fluorescent background.
An alternative highly quenched version of this assay exists, which uses a quenched fluorescent substrate. In this case, substrate degradation leads to loss of quenching and increased fluorescence in areas with enzyme activity.
Advantages: this technique provides information about endogenous gelatinase activity in situ. It can detect net enzyme activities because inactive pro-forms and enzyme-inhibitor complexes are not disrupted. It is relatively low cost and easy to perform.
Considerations: in situ zymography cannot be used to obtain qualitative data and is relatively low throughput. The use of artificial peptides may not report true in vivo activity if differences between the peptide and the endogenous substrate affect enzyme kinetics.
MMP-2 and MMP-9 share similar peptide recognition sequences; therefore, reported activity is likely to result from MMP-2 and MMP-9 activity combined. Separating the activity of these two individual enzymes may be achieved by using in situ hybridization in parallel, as long as the enzymes have different localization, or by using specific inhibitors.
When to use: use when you require non-quantitative, localization data.
Sample types: living tissue and whole organisms.
How it works: fluorescent substrate, such as modified type IV collagen, is injected into the tissue. The substrate either contains a fluorophore and a quencher, or is highly quenched, resulting in increased fluorescence upon substrate hydrolysis.
Advantages: gives real-time, 3D information about MMP-9 activity. Artifacts associated with sample preparation are minimized.
Considerations: in vivo zymography provides qualitative, rather than quantitative data. As with other techniques, the use of artificial peptides may not report true in vivo activity.
When to use: to see patterns of MMP-9 activity in vivo.
1. Hadler-Olsen E, Kanapathippillai P, Berg E, Svineng G, Winberg J-O, Uhlin-Hansen L. Gelatin in situ zymography on fixed, paraffin-embedded tissue: zinc and thanol fixation preserve enzyme activity. J Histochem Cytochem 58 29–39 (2010).
2. Toth M and Fridman R. Assessment of gelatinases (MMP-2 and MMP-9) by gelatin zymography. Methods Mol Med 57, 10 (2001).
3. Leber TM, Balkwill FR. Zymography: a single-step staining method for quantitation of proteolytic activity on substrate gels. Anal Biochem 249, 24–28 (1997).
4. Rossano R, Larocca M, Riviello L, Coniglio MG, Vandooren J, Liuzzi GM, Opdenakker G, Riccio P. Heterogeneity of serum gelatinases MMP-2 and MMP-9 isoforms and charge variants. J Cell Mol Med 18, 242–252.
5. Ratikov B, Deryugina E, Leng J, Marchenko G, Dembrow D, Strongin A. Determination of matrix metalloproteinase activity using biotinylated gelatin. Anal Biochem 286, 149–155 (2000).
6. Baragi VM, Shaw BJ, Renkiewicz RR, Kuipers PJ, Welgus HG, Mathrubutham M et al. A versatile assay for gelatinases using succinylated gelatin. Matrix Biol 19, 267–273 (2000).
7. Grierson C, Miller D, LaPan P, Brady J. Utility of combining MMP-9 enzyme-linked immunosorbent assay and MMP-9 activity assay data to monitor plasma enzyme specific activity. Anal Biochem 404, 232–4 (2010).
8. Vandooren J, Geurts N, Mertens E, Van den Steen PE, Opdenakker G. Zymography methods for visualizing hydrolytic enzymes. Nat Methods 10, 211–20 (2013).
9. Lauer-Fields J, Sritharan T, Stack, MS, Nagase H, Fields GB. Selective hydrolysis of triple-helical substrates by matrix metalloproteinase-2 and -9. J Biol Chem 278, 18140–18145 (2003).
10. Hawkins KE, DeMars KM, Yang C, Rosenberg GA, Candelario-Jalil E. Fluorometric immunocapture assay for the specific measurement of matrix metalloproteinase-9 activity in biological samples: application to brain and plasma from rats with ischemic stroke. Mol Brain 6, 14 (2013).